In the human endometrium, the mitotic effect of estrogen on epithelial cells is, normally mediated by paracrine factors that are secreted by endometrial stromal cells and act on the epithelial cells. Despite this, Selective Estrogen Receptor Modulators (SERMs) and protective, anti-proliferative phytoestrogens that influence this process have only been characterized in malignant epithelial cell lines where this indirect control mechanism is absent. Insulin-like Growth Factor (IGF) is thought to be the principal paracrine factor secreted by endometrial stromal cells that induces the proliferation of endometrial epithelial cells. We hypothesize that estrogens regulate secretion of paracrine factors, yielding the uterotrophic effect of tamoxifen or antiestrogenic protective effects of phytoestrogens in vivo, by differential signaling via estrogen receptors (ER) alpha and beta in stromal cells. The modulating effects of estrogen on epithelial proliferation could be explained either directly by inhibition of IGF synthesis in stromal cells, or indirectly by changing the ratio of the IGF Binding Proteins in stromal cells. To facilitate investigation of these mechanisms we recently developed strains and lines of human endometrial stromal cells immortalized by transduction of the telomerase reverse transcriptase gene. Using these cells and specific inhibitors of ER alpha and ER beta we will investigate the respective roles of the two receptors in regulating the synthesis of paracrine factors. In this revised application, factors secreted by stromal cell lines, particularly the IGF family of peptides, will be assessed under the influence of estrogen agonists and antagonists, in the presence or absence of specific ER-alpha or ER-beta inhibitors. This will test whether estrogen signaling either through ER alpha or ER beta produces different profiles of IGF pathway components which explain differences in the mitotic rates of endometrial epithelial cells. Conditioned medium, produced by stromal cells following stimulation by these estrogens will be tested for mitogenic activity on Ishikawa endometrial cancer cells and primary normal endometrial epithelial cell cultures. To prove their roles, we will test conditioned medium where IGF components and other factors have been depleted by immunoprecipitation. This study will test whether the hypothesized mechanism explains the differing responses of endometrium to estrogens and may allow development of a better assay of effects of SERMs and phytoestrogens on endometrium.